A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway.

Abstract

A coding mutation in the human insulin gene (His-B10 -* Asp) is associated with familial hyperproinsulinemia. To model this syndrome, we have produced transgenic mice that express high levels of the mutant prohormone in their islets of Langerhans. Strain 24-6 mice, containing about 100 copies of the mutant gene, were normoglycemic but had marked increases of serum human proinsulin immunoreactive components. Biosynthetic studies on isolated islets revealed that =65% of the proinsulin synthesized in these mice was the human mutant form. Unlike the normal endogenous mouse proinsulin, which was almost exclusively handled via a regulated secretory pathway, up to 15% of the human [Aspl'proinsulin was rapidly secreted after synthesis via an unregulated or constitutive pathway, and =20% was degraded within the islet cells. The secreted human [Aspl'lproinsulin was not processed proteolytically. However, the processing ofthe normal mouse and human mutant proinsulins within the islets from transgenic mice was not sigiicantly impaired. These findings suggest that the hyperproinsulinemia of the patients is the result of the continuous secretion of unprocessed mutant prohormone from the islets via this alternative unregulated pathway.In earlier studies we defined a mutation at position B10 (His Asp) in the coding region of an insulin allele that cosegregated with affected members of a kindred with hyperproinsulinemia (1, 2). The predicted amino acid substitution does not involve the dibasic processing sites for conversion of proinsulin to insulin, as has been found in two other families with hyperproinsulinemia (3, 4). However, replacement of the histidine residue at position B10 with aspartic acid would be expected to inhibit the association of this proinsulin into hexamers in the presence of zinc, inasmuch as coordination of zinc by this residue stabilizes insulin hexamers (5). The isoelectric point of the mutant (pro)insulin is reduced, while its receptor binding affinity and biological potency are increased by about 4.5-fold (6). Although none of these changes in the properties ofthe [Asplg0proinsulin would necessarily be expected to alter its proteolytic processing to insulin, the clinical phenotype of this mutation is one of greatly increased circulating proinsulin levels with mild carbohydrate intolerance (1).To explore the mechanism leading to the hypersecretion of proinsulin in these patients, we have introduced the mutant human insulin gene into mouse embryos to create transgenic animals in which the abnormal proinsulin is expressed in the islets of Langerhans along with the two normal mouse proinsulins. The results indicate that the mutant proinsulin is selectively secreted in increased amounts via an unregulated pathway, bypassing proteolytic conversion and leading to hyperproinsulinemia. METHODSProduction of Transgenic Mice. A 12.7-kilobase (kb) HindIII DNA fragment containing the human [AspBlOIinsulin gene was isolated from recombinant clone AMD41 by electrophoretic elution from an agarose gel (7). Ferti...

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

“…Wild-type human insulin shows a sorting index of 0.66 (Powell et al, 1988). Thus, the dimeric insulin B10Asp shows a slightly reduced efficiency of sorting, consistent with earlier published results (Carroll et al, 1988;Gross et al, 1989). Monomeric insulin B9Asp shows a sorting index of 0.67 + 0.02 and therefore is sorted as efficiently as wild-type insulin.…”

“…Taken together, the data showed that both mutated proinsulins entered the regulated secretory pathway where they are proteolytically processed and localized to densecore secretory granules. The B10Asp data are consistent with those of Carroll et al (1988) and Gross et al (1989) who showed that a significant fraction of the B10Asp was processed into mature insulin within the cells, presumably in dense-core secretory granules.…”

“…Our analysis of this mutant also showed a modest decrease in the efficiency of sorting: B10Asp is sorted at approximately two-thirds the efficiency of wild-type insulin (sorting index of 0.40 compared to 0.66). This level of diversion is comparable to that found in transgenic mice and in AtT-20 cells: Carroll et al (1988) showed that only 85% of the mutant prohormones were sorted into storage granules in the pancreas of transgenic mice compared to 97 % for the wild-type protein; Gross et al (1989) showed that in AtT-20 cells, 90% of labeled wild-type insulin was retained within the cells after a 30-min chase period whereas only 70% of B10Asp was retained. In all the studies, it is clear that a significant portion of the mutant insulin is still targeted to the storage granules and released by the regulated pathway.…”

“…The histidine residue at position B10 is involved in coordinating zinc ions (Blundell et al, 1972), and in the mutated form insulin dimers are formed instead of hexamers (Brange et al, 1988). Therefore, an intriguing possibility is that the aggregation state of insulin is important for its correct sorting into the regulated secretory pathway (Carroll et al, 1988;Gross et al, 1989). In this report, we set out to examine whether the intracellular transport and sorting of insulin is affected by its quaternary structure.…”

“…In a kindred with this disorder (Gruppuso et al, 1984), the hyperproinsulinemia was found to be associated with a point mutation in the B-chain coding region of the insulin gene (Chan et al, 1987). Carroll et al (1988) and Gross et al (1989) have examined the cellular basis for the defects of this mutant in transgenic mice and in transfected AtT-20 cells, respectively. In both cases, the defects appear to result from a decrease in the efficiency of sorting of the mutant proinsulin, leading to heightened secretion of the unprocessed prohormone in an unregulated fashion.…”